Project Summary The intestinal microbiota is extremely important to human health and development. Over the last decade, there has been a tremendous surge in the number of studies analyzing the gut microbiota; however, there are still relatively few mechanistic studies aimed at understanding basic biological properties of this ecosystem and its members. In recent years, our lab has been studying how predominant gut Bacteroidales members interact with each other in both beneficial and competitive/antagonistic relationships. These studies are essential to understanding how these bacteria become established in the mammalian intestine to form stable health- promoting communities. This proposal is a continuation of our studies of antagonism among these bacteria, specifically studying secreted antimicrobial molecules produced by the gut Bacteroidales. Under the initial project, we made unexpected findings regarding the types of secreted antimicrobial molecules produced, the range of Bacteroidales species that secrete antimicrobial molecules, and the mechanisms of resistance in producing strains. The goal of this renewal project is to understand the breadth of secreted antimicrobial molecules produced by the gut Bacteroidales, their targets and mechanisms of action in sensitive cells, and their importance in mediating competition, invasion and defense in the mammalian gut microbiota. In Aim 1, we will study a large class of proteins with membrane attack/perforin domains produced by diverse gut Bacteroidales using predictive data to determine which of these molecules have antimicrobial activity and how they target sensitive cells. In Aim 2, we will study a eukaryotic-like ubiquitin molecule with antimicrobial activity, likely acquired by B. fragilis by inter-kingdom transfer, and determine its target and mechanism of action. Aim 3 is designed to analyze the breadth of antimicrobial molecules produced by the gut Bacteroidales by studying diverse Bacteroidales species that produce potent secreted antimicrobial molecules of different classes. In Aim 4, we will use a combination of analyses, including computational modeling, experimental analyses in gnotobiotic mice, and analyses of human metagenomic datasets to understand the ecological relevance of secreted antimicrobial molecules of the gut Bacteroidales and how they contribute to ecosystem invasion, defense, and stability. Currently, we are the only group studying secreted antimicrobial proteins of the gut Bacteroidales and their ecological implications. The comprehensive analyses that will be performed in this proposal are unprecedented in the field and the ecological properties that we uncover will serve as guiding principles that can be applied to numerous human health applications such as restoring a healthy microbiota to dysbiotic patients, and the creation of improved probiotics.